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. Author manuscript; available in PMC: 2021 Jul 26.
Published in final edited form as: Methods Mol Biol. 2021;2270:341–358. doi: 10.1007/978-1-0716-1237-8_19

B Cell Commitment to IL-10 Production: the VertX Il10egfp mouse

Akihiko Oka 1,2, Bo Liu 1, Jeremy W Herzog 1, R Balfour Sartor 1,3,4
PMCID: PMC8312906  NIHMSID: NIHMS1695474  PMID: 33479908

Abstract

Although the inflammatory cytokine IL-10 is pivotal in regulatory B cell function, detecting IL-10-producing B cells by intracellular IL-10 staining requires multiple steps and tedious preparation. In contrast, the Il10-eGFP-reporter mouse model (VertX), generated in 2009, allows easier and quicker detection of IL-10-producing B cells with the possibility of sorting viable cells without membrane permeabilization and ex vivo activation. Even though detecting IL-10+ cells is simpler, several nuances are important. For example, methanol-containing buffers delete GFP signal while long-term fixation can maintain GFP intensity but decreases other intracellular signals (FOXP3 etc.). Here, we provide optimized and improved protocols for GFP detection in intestinal B cells and isolation techniques of lamina propria, spleen, mesenteric lymph node, peritoneum and blood cells from VertX mice.

Keywords: Regulatory B cell, Interleukin-10, GFP, mucosal immunology, flow cytometry, microbiota, gnotobiotic mice

1. Introduction

B lymphocytes are able to both drive and inhibit inflammation. They contribute to maintain homeostasis by producing immunoglobulins and cytokines and through antigen-presentation [1, 2]. Interleukin (IL)-10 is a key anti-inflammatory cytokine produced by lymphocytes, macrophages, dendritic cells and epithelial cells [3]. Multiple studies suggest that IL-10-producing B cells play a pivotal role in maintaining mucosal homeostasis [1–4]. Dysfunctional or decreased IL-10-producing regulatory B cells have been observed in several immune-related diseases in human and rodent models [1, 5–9]. Resident microbiota and certain helminths can induce IL-10-producing B cells and attenuate inflammation [1, 2, 4, 10, 11]. Despite the critical protective role of IL-10-producing B cells, their dynamics and functional analyses remain elusive, although these are cells being studied with emerging interest [8, 10, 11].

Several techniques, including flow cytometry, immunohistochemistry, quantitative PCR and western blotting have been used to detect intracellular IL-10. Although flow cytometry is cell-specific and quite sensitive, it requires exacting and prolonged in vitro preparation (6–8 hours in general) before proceeding with the staining of the intracellular cytokine: 1) pharmacological cell stimulation with phorbol myristate acetate, ionomycin and lipopolysaccharide or other activators, 2) protein-transport-blocking with brefeldin A or monensin, 3) cell surface marker staining and 4) cell fixation followed by the permeabilization of the cell membrane. These multiple steps may potentially change physiologic cytokine levels and transcriptional profiles, and decrease cell recovery. Cytokine-reporter mice, characterized by the insertion of a reporter gene (green fluorescent protein [GFP] or other markers) into the target cytokine gene-promoter locus, constitute an improved tool to follow cytokine production since you avoid cell membrane permeabilization and skip long-term preparation. Moreover, by using cytokine-reporter mice, the cytokine-producing cells are studied in their ‘intact’ or native state. About ten years ago, Dr. Karp and co-workers created the Il10-eGFP-reporter mouse (VertX) with intact production of IL-10 [3], which is now commercially available from the Jackson Laboratory [12]. Further, germ-free (GF) VertX mice are available from the National Gnotobiotic Rodent Resource Center at the University of North Carolina at Chapel Hill (UNC-CH) [13].

This chapter describes the following methods with some critical notes for GFP detection: 1) cell isolation from spleen, mesenteric lymph node (MLN), intestinal lamina propria (LP), peritoneum and peripheral blood mononuclear cells of VertX mice, 2) surface and intracellular staining for flow cytometry and flow-sorting of GFP (IL-10)+ B cells and 3) representative functional analyses using VertX mice.

2. Materials

2.1. Mice

  1. Specific-pathogen-free (SPF) Il10-eGFP-reporter (VertX, Il10egfp) mice: provided by UNC-CH, were originally obtained from Dr. Karp [3]. This strain is commercially available from the Jackson Laboratory (B6(Cg)-Il10tm1.1Karp/J) [12]. The genetic background is C57BL/6J.

  2. GF VertX mice: provided by the National Gnotobiotic Rodent Resource Center at UNC-CH [13] where the GF strain was established by embryo transfer. The genetic background is C57BL/6J.

  3. SPF VertX;PI3KδD910A mice: the phosphoinositide 3-kinase catalytic subunit p110δ (PI3Kδ) plays a critical role in IL-10 production by B cells [10, 11]. PI3Kp110δD910A/D910A mutant (PI3KδD910A) mice on the C57BL/6J background, which have a loss-of-function (catalytically inactive) point mutation in a gene encoding the PI3K catalytic subunit p110δ, were previously obtained from Dr. Vanhaesebroeck [14]. VertX;PI3KδD910A mice on a C57BL/6J background were generated by crossing PI3KδD910A with VertX mice at UNC-CH.

  4. SPF C57BL/6J mice: bred at UNC-CH, were originally obtained from the Jackson Laboratory [15].

2.2. Equipment

  1. Refrigerated centrifuge, unfixed angle centrifuge.

  2. Autoclave.

  3. CO2 incubator.

  4. Multi-position magnetic stirrer and stirrer bar.

  5. Devices for immunomagnetic separation (magnet and stand, columns, 30 μm pre-separation filters).

  6. Automated cell counter.

  7. Instruments for organ harvest: forceps, scissors, paper, petri dish and quad dish.

  8. 50 ml glass beaker (or 50 ml polypropylene autoclavable beaker).

  9. 15 ml and 50 ml conical tubes and 1.5 ml microcentrifuge tubes.

  10. Flow cytometry: an analyzer with up to 4 laser wavelengths for laser excitation (e.g. BD LSR II cell analyzer), a cell sorter (e.g. FACSAria III cell sorter with FACSDiva software, version 8, BD) and a flow cytometry data analysis tool (FlowJo analysis, version 10, FlowJo).

  11. 96 deep well 1 ml plate.

  12. Microplate shaker.

  13. Vortex.

  14. 20 G needle with cap, sterile.

  15. Animal lancet, 4 mm point length, sterile.

  16. 1 ml and 10 ml syringe, sterile.

  17. 70 μm and 100 μm cell strainer.

2.3. Buffers and reagents for cell isolation from different tissues

  1. 10x Phosphate-buffered saline (PBS): 80 g NaCl, 2 g KCl, 17.8 g Na2HPO4·2H2O, 2.4 g KH2PO4, adjust final volume to 1000 ml with distilled water, sterilize by autoclaving, pH 6.8 (see Note 1).

  2. Hank’s balanced salt solution (HBSS): 8 g NaCl, 400 mg KCl, 140 mg CaCl2, 100 mg MgSO4·7H2O, 100 mg MgCl2·6H2O, 60 mg Na2HPO2·2H2O, 60 mg KH2PO4, 1 g D-Glucose, 350 mg NaHCO3, adjust final volume to 1000 ml, sterilize by autoclaving, pH 7.4.

  3. Harvest buffer: 50 μg/ml gentamycin in 1x PBS.

  4. Wash medium: 2.5% heat-inactivated fetal bovine serum (HI-FBS) and 100 U/ml penicillin-streptomycin in HBSS.

  5. Red blood cell lysing buffer: 150 mM ammonium chloride, 10 mM potassium bicarbonate and 0.1 mM EDTA (see Note 2).

  6. Epithelial removal medium: 1 mM DL-dithiothreitol, 1 mM EDTA, 2.5% HI-FBS and 100 U/ml penicillin-streptomycin in HBSS.

  7. Digestion medium: 0.5 mg/ml collagenase Type IV, 2.5% HI-FBS and 100 U/ml penicillin-streptomycin in HBSS.

  8. 100% Percoll (prepare 100 ml): 90% of Percoll (90 ml) + 10% of 10x PBS (10 ml).

  9. 40% Percoll in HBSS (prepare 100 ml): 40% of 100% Percoll (40 ml) + 60% of HBSS (60 ml) (see Note 3).

  10. 70% Percoll in HBSS (prepare 100 ml) = 70% of 100% Percoll (70 ml) + 30% of HBSS (30 ml) (see Note 3).

  11. Heparin (stock 173 USP units/mg).

  12. Anti-CD19 magnetic beads (see Note 4).

  13. Modified MACS buffer: 10% HI-FBS in 1x PBS.

2.4. Regents for the identification of GFP+ B cells by flow cytometry

  1. Antibodies/reagents for cell staining (see Table 1). Every antibody/reagent needs to be titrated starting from manufacturer’s instructions.

  2. Compensation bead kit.

  3. Fixation buffer: 4% paraformaldehyde and 0.01% Tween 20 in 1x PBS.

  4. Phorbol 12-myristate 13-acetate (PMA, stock 1 mg/ml).

  5. Ionomycin (stock 1 mg/ml).

  6. GolgiStop (0.26% protein transport inhibitor monensin, BD).

  7. Complete culture medium: 10% HI-FBS, 100 U/ml penicillin/streptomycin, 1 mM sodium pyruvate and 50 μM 2-mercaptoethanol in RPMI1640.

  8. Permeabilization buffer: 0.1% Triton X-100, 0.5% bovine serum albumin (BSA) and 2 mM EDTA in 1x PBS.

  9. CpG-ODN 1826 (stock 500 μM) (see Note 5).

Table 1.

Antibodies/reagents for surface staining

Antibody/reagent Clone Fluorochrome Isotype
Fc Blocker (anti-CD16/CD32) 2.4G2 – –
LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit – APC-Cy7 –
CD45 30-F11 Pacific Orange Rat IgG2b κ
B220 (CD45R) RA3–6B2 Pacific Blue Rat IgG2a κ
CD19 6D5 BV605 Rat IgG2a κ
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3. Methods

To date, several IL-10 reporter strains have been published (see Fig. 1) [3, 16–21] and nicely summarized by Bouabe [22]. The VertX strain was generated by inserting an internal ribosome entry site (IRES)-eGFP cassette along with polyadenylation sequence of bovine growth hormone (BGHpA) at the end of the last exon of the IL-10 gene [3, 22] (see Fig. 1). In contrast to some other reporter strains, such as Il10eYFP, the IRES sequence enables the intact transcription of IL-10 along with eGFP under the control of the endogenous Il10 promoter. Il10 translation remains cap-dependent, whereas the translation of eGFP is driven by IRES [3, 22]. Furthermore, VertX strain has an advantage on stability of eGFP reporter mRNA because of the exchange of an endogenous mRNA-destabilizing 3’ untranslated region (3’UTR) for an exogenous mRNA-stabilizing polyadenylation sequence (BGHpA). In addition, BGHpA has higher mRNA and protein expression level than other polyadenylation sequences, such as simian vacuolating virus 40 (SV40pA) used in the other reporter strains [22]. Therefore, in VertX mice, functionally intact IL-10 protein can be secreted rapidly, whereas the GFP reporter protein remains intracellular longer [3, 22]. VertX mice indeed show higher reporter sensitivity compared to other reporter strains: many types of cells including B cells express eGFP reporter in VertX mice, whereas in some other reporter strains, only CD4+ T cells express reporter in steady state [3, 22]. In this section, we describe cell isolation and GFP detection methods suitable for VertX mice.

Figure 1. Il10-reporter allele in the Il10 locus of reporter mice.

Figure 1.

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3’UTR: 3’ untranslated region. eYFP: enhanced yellow fluorescent protein. IRES: internal ribosome entry site. eGFP: enhanced green fluorescent protein. BGHpA: bovine growth hormone polyadenylation sequence. SV40pA: simian vacuolating virus 40 polyadenylation sequence. Thy1.1: Thymus cell antigen 1.1 (CD90.1). Bla: β-lactamase (reporter enzyme). References: Il10eYFP mice [16], tiger mice [17], B-Green mice [20], ITIG mice [21], VertX mice [3], Il10Venus mice [19], 10BiT mice [18] and ITIB mice [21]. Figure is adapted and modified from [22].

3.1. Isolation of cells from several tissues

The following protocols describe the procedures employed to isolate the total cell population from different organs. Regardless of the tissue under analysis, the subsequent purification of wild type and of GFP+ B lymphocytes from the total pool of cells is performed by positive selection using anti-CD19 magnetic beads and following manufacturer’s instructions (see Note 4). Table 2 shows yields of both total and B cells obtained through these isolation protocols.

Table 2. Yield of cell isolation.

(×106 cells/mouse, mean ± SD)

MLN
 Spleen
Mice Total CD19+ Total CD19+
GF VertX 12 ± 4.1 1.9 ± 0.4 73 ± 33 21 ± 5.9
SPF-colonized exGF VertX 45 ± 8.4 5.0 ± 1.5 184 ± 49 35 ± 7.2
SPF-raised VertX 16 ± 4.4 2.6 ± 0.5 86 ± 21 24 ± 8.6

cLP
 Peritoneum
 PBMC
Total CD19+ Total Total
2.7 ± 1.4 0.4 ± 0.2 N/A N/A
5.3 ± 4.8 1.0 ± 0.2 N/A N/A
4.1 ± 2.2 0.5 ± 0.3 6.5 ± 0.5 1.0 ± 0.5
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GF: germ-free, SPF: specific-pathogen-free, ex-GF: germ-free mice colonized for 2 weeks, MLN: mesenteric lymph node, cLP: colonic lamina propria, PBMC: peripheral blood mononuclear cell, N/A: not available, N=3–37 mice/group, 8–14 weeks old.

3.1.1. Isolation of spleen and mesenteric lymph node cells

  1. Harvest spleen and mesenteric lymph nodes (MLN) without fat tissue and place in cold harvest buffer.

  2. Smash the spleen and MLN by needle cap (or rubber plunger head) on 70 μm cell strainer and wash the strainer with 5 ml of cold wash media (see Fig. 2). Centrifuge at 450 x g for 5 min at 4°C and discard supernatant.

  3. For splenocytes (for MLN cells, go to step 4), resuspend pellet in red blood cell lysing buffer (5 ml/spleen) and incubate for 3–5 min at 18–22°C. Add 10 ml of wash medium and filter through 70 μm cell strainer. Centrifuge at 450 x g for 5 min at 4°C and discard supernatant. If the cell pellet looks still red, repeat step 3.

  4. Resuspend cells in complete culture medium and count cells.

Figure 2. Isolation of spleen and mesenteric lymph node cells.

Figure 2.

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In order to obtain a single cell population, spleens or mesenteric lymph nodes are smashed on a cell strainer by the needle cap (or rubber plunger head).

3.1.2. Isolation of intestinal lamina propria mononuclear cells

This protocol is suitable for colonic lamina propria. For small intestine, duodenum and stomach, increase EDTA final concentration in epithelial removal medium from 1 mM to at least 3 mM.

  1. To prepare the tissue, harvest colon without fat tissue and place in cold harvest buffer (see Note 6). Cut intestine longitudinally to remove luminal contents and wash in cold harvest buffer (see Fig. 3a). Remove mucus by rolling intestine on paper towel (see Note 7).

  2. To remove the epithelial layer, cut intestine into 10 mm pieces and incubate in 20 ml of pre-warmed (37°C) epithelial removal medium for 20 min at 37°C with 250 rpm stir in 50 ml beaker (see Fig. 3b). Discard supernatant by aspiration with pipet (or pouring) and wash the intestine with pre-warmed (37°C) wash medium to remove DL-dithiothreitol and EDTA (see Note 8).

  3. To digest the lamina propria, incubate intestine in 20 ml of pre-warmed (37°C) digestion medium for 30 min at 37°C with 350 rpm stir in 50 ml beaker (see Note 9). Filter supernatant (containing lamina propria cells) and digested intestine through a 100 μm cell strainer with pre-warmed (37°C) wash medium. Centrifuge at 450 x g for 10 min at 18–22°C and discard the supernatant.

  4. Resuspend the pellet in 5 ml of 40% Percoll in 15 ml conical tube. Carefully add 5 ml of 70% Percoll under the 40% Percoll layer with a 5 ml pipet (or dropper) without disrupting the 40/70% interface. Centrifuge at 800 x g for 20 min at 18–22°C with slow acceleration and brake off. At the end of the centrifugation, collect the white layer (leukocyte layer) in the 40/70% interface with a 1000 μl tip (or dropper) (see Fig. 3c) (see Note 10). Resuspend in 50 ml of cold HBSS and centrifuge at 450 x g for 10 min at 4°C and discard the supernatant. Resuspend cells in complete culture medium and count cells.

Figure 3. Lamina propria cell isolation.

Figure 3.

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(a) The colon is washed in a quad dish. (b) Incubation of colon pieces in beaker with stirrer bar. (c) Result of the Percoll density gradient centrifugation.

3.1.3. Isolation of Peritoneal Cells

  1. Inject 10 ml of RPMI medium into the peritoneal cavity by 10 ml syringe with a 20 G needle. Tilt the mouse vertically and horizontally and collect the injected RPMI medium by a 10 ml syringe with a 20 G needle.

  2. Filter through a 70 μm cell strainer with wash medium. Centrifuge at 450 x g for 10 min at 4°C and discard supernatant. Resuspend cells in complete culture medium and count cells.

3.1.4. Isolation of peripheral blood mononuclear cells

  1. Collect 500 μl of blood by cheek puncture with animal lancet or postmortem cardiac puncture into a heparin-coated 1.5 ml tube.

  2. Dilute blood 1:1 with HBSS and then put 1 ml of the diluted blood onto 1 ml of 60% Percoll in HBSS for density gradient centrifugation. Centrifuge at 800 x g for 10 min at 18–22°C with slow acceleration and brake off. Collect the white mononuclear cell layer at the interface of plasma/Percoll and resuspend in 15 ml of cold HBSS. Centrifuge at 450 x g for 10 min at 4°C and discard the supernatant. Resuspend cells in complete culture medium and count cells.

3.2. Identification of GFP+ B cells by flow cytometry

Because the GFP+ population is frequently small in VertX mice with dim GFP signal that can be masked with auto-fluorescence, precise negative and positive controls are required. A negative control for GFP to create appropriate positive and negative gates requires a fluorescence-minus-one (FMO) control, which is represented by non-eGFP-reporter cells incubated with the same antibodies used to stain the experimental samples (see Fig. 4a) [23]. Importantly, some fixation and permeabilization buffers, especially the ones containing methanol, dampen GFP intensity (see Fig. 4b) [24], while the buffers described in this protocol do not

Figure 4. GFP (IL-10) gating strategy.

Figure 4.

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(a) Colonic lamina propria cells isolated from VertX mice were stained (LIVE/DEAD and anti-CD45, -B220 and -CD19 antibodies) and analyzed by flow cytometry. For the fluorescent-minus-one (FMO) control, non-eGFP-reporter cells from the colonic lamina propria of C57BL6/J mice were stained by all antibodies and used to set the GFP positive gate. (b) Splenocytes isolated from VertX mice were cultured with 500 nM CpG-ODN in complete culture medium for 24 h. The cultured splenocytes were stained with the LIVE/DEAD dye and anti-CD45 antibody and analyzed by flow cytometry. The flow dot plot shows singlet-live-CD45+ splenocytes before exposure to methanol-containing buffer (left panel) and after incubation with methanol-containing buffer for 5 min (right panel). A reduction of GFP (IL-10) signal was observed when using a methanol-containing buffer.

3.2.1. Staining of surface markers for flow cytometry

  1. Prepare samples by resuspending 1 x 106 cells in 45 μl of 1x PBS in a 96 well plate (see Note 11). The FMO control (non-eGFP-reporter cells from C57BL/6J mice) should be included as well as samples in the experimental plan (see Note 12).

  2. For cell surface staining, incubate cells for 5 min at 4°C with Fc blocker diluted according to manufacturer’s instructions. Add 50 μl of titrated antibodies diluted in 1x PBS (see Table 1) and incubate at 4°C for 20 min. After staining, add 100 μl of 1x PBS, centrifuge at 350 x g at 4°C for 5 min and flick off supernatant.

  3. To fix the samples, resuspend cells in 50 μl of pre-warmed (37°C) fixation buffer and incubate at 37°C for 5 min (see Note 13). Add 150 μl of 1x PBS, centrifuge at 350 x g for 5 min and flick off supernatant.

  4. Resuspend cells in 200 μl of 1x PBS and keep at 4°C before flow cytometry analysis. Representative results using this protocol are shown in Fig. 4.

3.2.2. Staining intracellular cytokines for flow cytometry

GFP-reporter cells do not require permeabilization. If other cytokines (TGF-β1 etc.) or transcriptional factors (FOXP3 etc.) are stained, proceed with the following steps.

  1. After isolating cells from tissues, culture cells at 1–5 x 106 cells/ml with 50 ng/ml PMA and 1 μg/ml ionomycin in complete culture medium for 4 h with vortexing every 30 min. Add GolgiStop (1 μl/ml) together for the last 3 h. After 4 h incubation, add 10 ml of 1x PBS, centrifuge at 350 x g for 5 min and discard supernatant.

  2. For sample preparation, resuspend 1 x 106 cells in 45 μl of 1x PBS buffer in a 96 well plate (see Note 11). FMO control (non-eGFP-reporter cells from C57BL/6J) should be included as well as samples (see Note 12).

  3. For cell surface staining, incubate cells for 5 min at 4°C with Fc blocker diluted according to manufacturer’s instructions. Add 50 μl of titrated antibodies diluted in 1x PBS, and incubate at 4°C for 20 min. After staining, add 100 μl of 1x PBS, centrifuge at 350 x g at 4°C for 5 min and flick off supernatant.

  4. To fix cells, resuspend cells in 50 μl of pre-warmed (37°C) fixation buffer and incubate at 37°C for 5 min (see Note 13). Add 150 μl of 1x PBS, centrifuge at 350 x g for 5 min and flick off supernatant.

  5. For the permeabilization step, incubate 1 x 106 cells in 50 μl of permeabilization buffer at room temperature for 45 min with shaking at 360 rpm. Add 150 μl of 1x PBS, centrifuge at 350 x g for 5 min and flick off supernatant.

  6. For intracellular staining, add 100 μl of titrated antibodies diluted in permeabilization buffer, and incubate overnight (or at least for 2 h). After intracellular staining, add 150 μl of 1x PBS, centrifuge at 350 x g for 5 min and flick off supernatant.

  7. Resuspend cells in 200 μl of 1x PBS for flow cytometry analysis.

3.2.3. Special compensation controls for flow cytometry

For flow cytometry compensation, we use the bead kit which provides clearly separated positive and negative signals in contrast to actual cells. However, GFP and LIVE/DEAD dye compensations need actual cells. Although GFP can be detected by the FITC channel, GFP and FITC have different emission spectra. Therefore, we need GFP-expressing cells instead of FITC-antibody-stained cells. In order to obtain strong GFP signal, it’s better to re-stimulate cells ex vivo with CpG-ODN because GFP expression in VertX mice is not so bright (dim) in a steady state. Similarly, as the LIVE/DEAD dye works for compromised membranes of apoptotic/necrotic cells (see Note 14), it is better to boil control cells to have strong dead signals.

  1. For the GFP positive control, culture overnight (or at least for 5 h) 1 x 106 cells from VertX with 500 nM CpG-ODN in complete culture medium in a 37°C and 5% CO2 incubator. Wash with 1x PBS and centrifuge at 350 x g for 5 min. Resuspend cells in 200 μl of 1x PBS for flow cytometry analysis (see Note 15).

  2. For the LIVE/DEAD staining positive control, boil 1 x 106 non-eGFP-reporter cells in hot water for 10 sec and stain with LIVE/DEAD staining dye in 1x PBS for 20 min. Wash with 1x PBS and centrifuge at 350 x g for 5 min. Resuspend cells in 200 μl of 1x PBS for flow cytometry analysis (see Notes 14 and 16).

3.3. Functional analysis of GFP+ B cell in vivo and in vitro

Here below, two representative methods which require the employment of VertX mice are described.

3.3.1. GFP+ B cells in exGF VertX mice

  1. Collect fresh feces from specific pathogen-free (SPF) C57BL/6J mice and pool together 3 feces in 3 ml of 1x PBS. Homogenize feces and inoculate to germ-free (GF) VertX mice by swabbing orally and rectally (conventionalization).

  2. By 2 weeks after conventionalization, detect GFP+ B cells in the lamina propria. Isolate cells as in Subheading 3.1.2 and proceed with cell surface staining as in Subheading 3.2.1. Cells can be then sorted or simply analyzed by flow cytometry. Fig. 5a shows the results of GFP (IL-10) induction in lamina propria B cells by resident microbiota colonization.

  3. For the flow-sorting of GFP+ B cells, create GFP positive and negative gates based on the FMO control (see Subheading 3.2). According to the sequential assay (culture, RNA sequencing, quantitative PCR etc.), sort cells directly into medium, TRIzol or RNAlater etc. (when required by the case, add RNase inhibitor) [25] with a 4 way-purity mode. Fig. 5b shows the results of post-sorting.

Figure 5. GFP+CD19+ cells induced by specific-pathogen-free (SPF) gut microbiota colonization in germ-free (GF) mice.

Figure 5.

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Two weeks after colonization, colonic lamina propria cells isolated from non-eGFP-reporter (C57BL6/J, as a fluorescent-minus-one [FMO] control) and VertX mice were stained (LIVE/DEAD dye and anti-CD45, -B220 and -CD19 antibodies) and flow-sorted in the 4-way-purity mode. GFP gates were set based on the FMO control. (a) Colonic lamina propria GFP+CD19+ cells in GF and SPF-colonized GF VertX mice. (b) Post-sort analysis showing the purity of flow-sorted cells.

3.3.2. Comparison of GFP expression between wild type and gene-modified B cells

Several genes are involved in IL-10 signaling. If certain gene is modified in GFP reporter mice, you can see reduced or increased GFP expression in the gene-modified mice. For example, PI3Kδ signaling positively regulates IL-10 signaling [10, 11].

  1. Isolate total splenocytes (or MLN cells) from VertX and VertX;PI3KδD910A mice. After counting cells, culture viable cells at 1 x 106 cells/ml in complete culture medium with 500 nM CpG-ODN. Incubate in a 37°C 5% CO2 incubator for 48 h (see Fig. 6a).

  2. At the end of the 48-h stimulation, collect the cells and stain with antibodies directed against the surface markers CD45, B220 and CD19 as described in Subheading 3.2.1.

  3. Run flow cytometry and compare GFP (IL-10) expression in B cells from VertX and VertX;PI3KδD910A mice. Fig. 6b shows reduced GFP (IL-10) production by PI3KδD910A mutation compared to PI3Kδ wild type.

Figure 6. GFP (IL-10) detection by flow cytometry.

Figure 6.

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(a) Unfractionated splenocytes isolated from non-eGFP-reporter (C57BL6/J, as a fluorescent-minus-one [FMO] control), VertX and VertX;PI3KδD910A mice were cultured with 500 nM CpG-ODN in complete media for 48 h. The cultured cells were stained (LIVE/DEAD dye and anti-CD45, -B220 and -CD19 antibodies) and analyzed by flow cytometry. GFP gates were set based on the FMO control. (b) Flow dot plots showing singlet-live-CD45+B220+CD19+ B cells and GFP positive (upper) and negative (lower) gates.

5. Acknowledgements

We thank Dr. C.L. Karp for providing VertX mice and Dr. B. Vanhaesebroeck for providing PI3KδD910A mice. The authors are supported by grants from National Institute of Health (P01DK094779; P30DK034987; P40OD010995) and Crohn’s & Colitis Foundation Research Fellowship Award (407007) and Gnotobiotic Facility. The UNC Flow Cytometry Core Facility is supported in part by P30CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center. The flow cytometry research reported in this publication was supported in part by the North Carolina Biotech Center Institutional Support Grant 2012-IDG-1006.

4 Notes

1.

The pH of the 10x stock will be 6.8. When diluted to 1x PBS, the pH should change to 7.4 which is the normal pH for 1x PBS. If necessary, adjust pH using hydrochloric acid or sodium hydroxide.

2.

The red blood cell lysing buffer can be homemade or purchased from different suppliers.

3.

To see interface clearly, HBSS for 40% Percoll is phenol-red (+) while HBSS for 70% Percoll is phenol-red-free.

4.

We routinely use CD19 microbeads from Miltenyi Biotec. The protocol for B cells and GFP+ B cells isolation is according to the manufacturer’s instruction, except for MACS buffer. Indeed, a modified MACS buffer was used (see Subheading 2.3).

5.

CpG ODN1826 sequence: 5’-TCCATGACGTTCCTGACGTT-3’ [26].

6.

Fat (adipose) tissue can reduce isolation yield and also potentially lead to contamination by immune cells present within the fat, such as macrophages, regulatory T cells, natural killer T cells, etc. [27].

7.

Mucus can reduce isolation yield due to clogging of the cell strainer.

8.

The supernatant contains epithelial layer cells, which can be used for other assays [28].

9.

To increase yield or shorter isolation time, additional collagenase, DNase I (40 μg/ml) or dispase (0.5–1 mg/ml) can be used [28–30]. Of note, these agents potentially disrupt lymphocyte surface markers likely due to contamination by proteases present in the enzyme preparations [31, 32]. Additional serum and trypsin inhibitor in digestion medium or overnight culture in complete culture medium after digestion may rescue this problem. Repeating short digestion rounds may help.

10.

Do not take much Percoll with white layer cells since it reduces the efficacy of sequential centrifugation. If there is no pellet after centrifugation, add more HBSS to the cell suspension to dilute Percoll and centrifuge again.

11.

Do not add FBS or BSA to the staining buffer when using LIVE/DEAD staining kit. The dyes in the kit react with proteins (FBS or BSA), resulting in no LIVE/DEAD signal. Use 1x PBS for staining buffer according to the manufacturer’s manual.

12.

For an optimal FMO control, use the same type of cells that means the same genetic background (C57BL6/J), same tissue, and same isolation process.

13.

Do not incubate longer than 5 min. Long-term fixation significantly reduces intracellular fluorescence signals [33].

14.

Another option is a compensation bead kit for LIVE/DEAD staining, the ArC Amine reactive compensation bead kit (Invitrogen), which does not require actual cells.

15.

As a GFP compensation control, another option instead of using freshly isolated cells is to defreeze GFP+ cells stored in RPMI1640 supplemented with 10% dimethyl sulfoxide and 10% HI-FBS. Wash thawed GFP+ cells with 1x PBS and resuspend 1 x 106 cells in 200 μl of 1x PBS for flow cytometry analysis.

16.

Do not permeabilize LIVE/DEAD stained cells. Permeabilization can reduce the intensity of the LIVE/DEAD dye.

6 References

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